MicroRNAs (miRNAs) are short, endogenous noncoding RNAs found in animals (Lee, R. C. et al., Cell 75:843-854 (1993); Wightman, B. et al., Cell 75:855-862 (1993); Lagos-Quintana, M. et al., Science 294:853-858 (2001); Lagos-Quintana, M. et al., Curr. Biol. 12:735-739 (2002); Lagos-Quintana, M. et al., RNA 9:175-179 (2003); Lau, N. C. et al., Science 294:858-862 (2001); Lee, R. C. et al., Science 294:862-864 (2001); Lim, L. P. et al., Science 299:1540 (2003a); Lim, L. P. et al., Genes Dev. 17:991-1008 (2003b); Mourelatos, Z., et al., Genes Dev. 16:720-728 (2002); Aravin, A. et al., Dev. Cell 5:337-350 (2003); Ambros, V. et al., Curr. Biol. 13:807-818 (2003a); Dostie, J. et al., RNA 9:180-186 (2003); Grad, Y. et al., Mol. Cell 11:1253-1263 (2003)), plants (Reinhart, B. J. et al., Genes Dev. 16:1616-1626 (2002); Llave, C. et al., Plant Cell 14:1605-1619 (2002a); Park, W. et al., Curr. Biol. 12:1484-1495 (2002); Mette, M. F. et al., Plant Physiol. 130:6-9 (2002); Palatnik, J. F. et al., Nature 425:257-263 (2003); Floyd, S. K. et al., Nature 428:485-486 (2004); Jones-Rhoades, M. J. et al., Mol. Cell 14:787-799 (2004); Sunkar, R. et al., Plant Cell 16:2001-2019 (2004); Wang, J-F. et al., Nucleic Acids Res. 32:1688-1695 (2004a); Wang, X. J. et al., Genome Biol. 5:R65 (2004b); Adai, A. et al., Genome Res. 15:78-91 (2005)), and the Epstein-Barr virus (Pfeffer, S. et al., Science 304:734-736 (2004)). In both animals and plants, the majority of the miRNA genes exists as independent transcriptional units and they are transcribed by RNA polymerase II into long primary transcripts (termed pri-miRNAs) (Bartel, D. P. Cell 116:281-297 (2004); Parizotto, E. A. et al., Genes Dev. 18:2237-2242 (2004); Kurihara, Y. et al., Proc. Natl. Acad. Sci. USA 101:12753-12758 (2004)). In animals, pri-miRNAs are trimmed in the nucleus to generate ˜70 nt miRNA precursors (pre-miRNAs) with fold-back structures by a multi-protein complex called microprocessor in which Drosha (an RNase III-like enzyme) and Pasha (a double-stranded RNA binding protein) are critical components (Lee, Y. et al., Nature 425:415-419 (2003); Denli, A. H. et al., Nature 432:231-234 (2004)). The pre-miRNAs are exported to the cytoplasm and subsequently cleaved by another RNase III-like enzyme called Dicer to generate mature miRNAs (Bernstein, E. et al., Nature 409:363-366 (2001)). However, the Arabidopsis genome does not appear to encode a Drosha ortholog, and it seems that the plant nuclear-localized Dicer homolog is likely to have Drosha function (Kurihara, Y. et al., Proc. Natl. Acad. Sci. USA 101:12753-12758 (2004)). Many miRNAs are conserved between species—often over wide evolutionary distances. For example, AthmiR166 is conserved in all lineages of land plants, including bryophytes, lycopods, ferns and seed plants (Floyd, S. K. et al., Nature 428:485-486 (2004)), and the Caenorhabditis elegans miRNA, let-7, is conserved in human, Drosophila, and eleven other bilateral animals (Pasquinelli, A. E. et al., Nature. 408:86-89 (2000)); but others are only conserved between more closely related species such as C. elegans and C. briggsae (Ambros, V. et al., Curr. Biol. 13:807-818 (2003a); Bartel, D. P. Cell 116:281-297 (2004)). miRNAs down-regulate the expression of specific mRNA targets, either by directing the cleavage of mRNAs or interfering with translation (Carrington, J. C. et al. Science 301:336-338 (2003); Bartel, D. P. Cell 116:281-297 (2004); Ambros, V. Nature 431:350-355 (2004)).
miRNAs have been identified by cloning and by computational approaches tailored to the key features of lin-4 and let-7, the 2 founding members of miRNAs from C. elegans, which include a fold-back hairpin RNA precursor coupled with evolutionary conservation (Ambros, V. et al., RNA 9:277-279 (2003b)). It was estimated that miRNA genes represent 1% of the expressed genome in complex organisms such as worms, flies and humans (Lai, E. C. Curr. Biol. 13:R925-R936 (2003); Lim, L. P. et al., Genes Dev. 17:991-1008 (2003b); Bartel, D. P. Cell 116:281-297 (2004)). However, recent computational predictions have raised the number of miRNAs significantly in primates by comparative analysis of the human, mouse and rat genomes (Berezikov, E. et al., Cell, 120:21-24 (2005)). The identification of the entire set of miRNAs and their target genes from model organisms is of fundamental importance to understand regulatory networks and gene silencing mechanisms.
Rice is the world's most important crop, as measured by the portion of calories it provides to the human diet. It is an established model system for monocots that include all cereals. Rice is the only monocot species with a fully sequenced genome. The availability of the complete genome sequence of rice allowed the in silico identification of 20 families of rice miRNAs based on conservation of sequences with Arabidopsis miRNAs (Reinhart, B. J. et al., Genes Dev. 16:1616-1626 (2002); Park, W. et al., Curr. Biol. 12:1484-1495 (2002); Jones-Rhoades, M. J. et al., Mol. Cell 14:787-799 (2004); Sunkar, R. et al., Plant Cell 16:2001-2019 (2004); Bonnet, E. et al., Proc. Natl. Acad. Sci. USA. 101:11511-11516 (2004); Wang, J-F. et al., Nucleic Acids Res. 32:1688-1695 (2004a); Adai, A. et al., Genome Res. 15:78-91 (2005)). In addition to finding conserved miRNAs, cloning approaches revealed Arabidopsis miRNAs that are not conserved in rice. At least four well-characterized Arabidopsis miRNAs, miR158, miR161, miR163 and miR173 do not have homologs in rice (Jones-Rhoades, M. J. et al., Mol. Cell 14:787-799 (2004)). Another miRNA (miR403) has been found to be conserved between Arabidopsis and Populus while its counterpart could not be identified in rice (Sunkar, R. et al., Plant Cell 16:2001-2019 (2004)). Recently, evidence was shown that the non-conserved miR161 and miR163 from Arabidopsis may have evolved by inverted duplication of their target genes (Allen, E. et al., Nature Genet. 36:1282-1290 (2004)). Additionally, Berezikov, E. et al., Cell, 120:21-24 (2005)) have predicted lineage specific miRNAs in mammalian and non-mammalian animal species. Taken together, these observations support the notion that rice may express monocot- and/or rice-specific miRNAs.
The present study was undertaken to identify new miRNAs that are difficult to predict in silico and verify previously predicted miRNAs from rice. Sequencing of small RNA libraries and subsequent analysis led to the identification of 14 new miRNAs. These new miRNAs from rice form 14 families, 13 of which are new and not present in Arabidopsis. Furthermore, we confirmed the existence of 15 of the 20 conserved families of miRNAs that were predicted previously. Based on sequence complementarity to miRNAs, we were able to predict 46 rice genes as putative targets of the new miRNAs. These predicted targets include not only transcription factors but also other genes involved in diverse physiological processes.